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X-S digest of KaiA/B and RFP BioBrick plasmid

We performed X-S digests of KaiA/B and the RFP BioBrick plasmids. There were 6 digests in total: LC3A KaiA, LC3A KaiB, LC3B KaiA, LC3B KaiB, RFP #3, and RFP #4 (RFP numbers refer to the 4 minipreps we did yesterday).


  • 20 µL DNA
  • 2.5 µL Buffer 2 (from NEB)
  • 0.25 µL BSA (100x)
  • 0.5 µL XbaI enzyme
  • 0.5 µl SpeI enzyme
  • 1.25 µL H2O

Incubate for 2 hours at 37C, heat inactivate for 20 minutes at 80C, hold at 4C.

Gel images from yesterday's mutagenic PCR

Click for legend

Gel images from yesterday's "all" PCR

Click for legend

Phosphotase treatment of KaiA/B and RFP BioBrick plasmid digestions

After completing the X-S digests, we added 1 µL alkaline phosphatase to each digestion and incubated for 1 hour at 37C.

Update: This was a mistake, I wasn't supposed to add phosphatase to KaiA and KaiB. We will redo the KaiA/B digests.

Reinoculation of PCC 7942 flask from frozen stock

Our PCC 7942 liquid cultures are now more than 2 weeks old, so we reinoculated a new flask from frozen stock so we have a fresh supply of cyanobacteria.

Gel purification of KaiA/B and RFP BioBrick plasmid

Click for legend

We gel-purified KaiA/B and the RFP BioBrick plasmids after digesting them and treating them with phosphotase. Gel image to the left.

Gel extraction of BioBrick backbone

We excised the 2kb fragments from lanes 6 and 7 above. They're sitting in the 4C refrigerator waiting to be purified.

Re-digest of KaiA/B and RFP BioBrick

We redigested LC3B KaiA and LC3B KaiB with X-S since I mistakenly added phosphatase to our earlier digests. Also, at Nick's request, we digested the RFP BioBrick again, with the intent of purifying the insert (not the backbone).